Crohn's disease is immunologically mediated with genetic and environmental components. Genetic factors include defective immunoregulation, mucosal barrier integrity/repair and bacterial killing, while enteric bacterial antigens stimulate intestinal inflammation. 4 genes associated with Crohn's disease (NOD 2, ATG 16L1, NCF4, IGRM) regulate bacterial killing, suggesting that defective innate responses can activate pathogenic adaptive immune responses to persistent bacteria. Commensal enteric bacteria antigen-specific effector T cells that cause chronic colitis in interleukin 10 deficient (IL-10-/-) mice. Selective colonization of germ-free IL-10-/- mice with Escherichia coli or Enterococcus faecalis induces different phenotypes of colitis for each bacteria. E. coli and E. faecalis strains exhibit diverse biologic activities, ranging from pathogenic to probiotic. Ileal Crohn's disease mucosa is colonized enteroadherent/ invasive E. coli that contain virulence genes that mediate invasion and intracellular persistence/ replication. The murine E. coli NC101 strain that causes colitis in our monoassociated IL-10-/- mice displays a similar pattern of genes. Differential abilities of E. coli and E. faecalis strains to cause colitis in monoassociated IL-10-/- mice correlate with the presence of several defined bacterial virulence factors. Hypothesis: Persistent exposure to invasive, translocating, intracellular commensal enteric bacteria induces aggressive antibacterial TH1 and TH17-mediated immune responses that cause chronic intestinal inflammation. Persistent bacterial exposure is caused by either: 1) Colonization with commensal strains that express virulence factors that promote intestinal epithelial cell adherence, invasion and resistance to intracellular killing within epithelial cells and macrophages, or 2) Genetically determined host innate defects in bacterial killing and mucosal barrier function. Individuals with genetically determined innate immune defects develop Crohn's disease when they are colonized with bacterial strains that possess these virulence factors. Specific Aims: 1. Determine whether E. coli and E. faecalis virulence genes that promote epithelial invasion, mucosal translocation and intracellular survival within macrophages and epithelial cells mediate experimental intestinal inflammation and mucosal immune responses. A. In vivo studies. Do bacterial genes that regulate: 1) mucosal invasion and translocation 2) intracellular survival or 3) secretion of oxygen radicals influence the ability of E. coli and E. faecalis strains to induce chronic T cell-mediated colitis in monoassociated IL-10-/- mice? 1. Described virulence genes. We will study E. coli genes with described in vitro functions and are found in both our colitogenic NC101 strain and CD isolates and E. faecalis genes with documented functional roles in other inflammatory conditions. 2. Novel virulence genes. Novel virulence factors in our murine colitogenic E. coli NC101 strain will be identified by sequencing NC 101, comparing known virulence genes with sequences of the human ileal CD AIEC isolate LF82, and the noncolitogenic E. coli K12, and identifying NC101 genes that determine epithelial invasion and translocation by signature tagged mutagenesis. B. In vitro studies. Does 1) intracellular bacterial survival and 2) secretion of superoxide by E. faecalis affect innate immune responses in dendritic cells and macrophages? 2. Determine the role of bacterial-induced NF:B signaling on innate bacterial killing by epithelial cells and macrophages exposed to invasive/persistent E. coli and E. faecalis strains. A. In vivo: Does epithelial NF:B signaling influence bacterial colonization, mucosal adherence, translocation and colitis in IL-10-/- mice? B. In vitro: Does TLR and NOD 2 activation of NF:B signaling influence in vitro epithelial barrier function, innate immune responses and epithelial and macrophage killing of various E. coli and E. faecalis strains? PUBLIC HEALTH RELEVANCE: Crohn's disease is a common chronic inflammatory condition of the intestine that has genetic and environmental components, although the exact cause is unknown. This application seeks to identify mechanisms by which 2 common nonpathogenic (normal) intestinal bacterial species can cause intestinal inflammation in genetically susceptible hosts. The bacterial genes explored in this proposal could be used as diagnostic tests for abnormal bacteria in Crohn's disease and understanding the mechanisms by which these bacteria induce chronic inflammation could provide targets for new medications to treat Crohn's disease.